Yuan Z M, Egorin M J, Rosen D M, Simon M A, Callery P S
Department of Biomedicinal Chemistry, University of Maryland School of Pharmacy, Baltimore 21201.
Cancer Res. 1994 Feb 1;54(3):742-8.
We have previously described the synthesis and cytotoxic properties of 2 polyamine analogues in which either the N1- or N8-amino group of spermidine was replaced by the alkylating moiety, aziridine. However, the mechanisms by which these aziridinyl analogues of spermidine inhibit cell growth remain unknown. As a result, we have studied: (a) the effect of pretreatment with difluoromethyl ornithine (DFMO) and coincubation with exogenous spermidine on cytotoxicity induced by the aziridinyl spermidines; (b) the reversibility of the cytotoxicity induced by the aziridinyl spermidines; (c) the accumulation of N1- and N8-aziridinyl spermidine by cells and the effects of DFMO on this process; and (d) the impact of N1- and N8-aziridinyl spermidine on cellular polyamine pools and on cellular accumulation of spermidine. The cytotoxicity induced by these 2 aziridinyl derivatives of spermidine [concentration required to inhibit cell growth or incorporation of radiolabeled precursor into trichloroacetic acid-precipitable material by 50% (IC50) N1 = 0.2 microM, IC50 N8 = 0.4 microM)] was potentiated by pretreatment of L1210 cells for 24 h with 100 microM DFMO (IC50 N1 = 0.05 microM, IC50 N8 = 0.15 microM) and was prevented by coincubation with 3.7 microM spermidine (IC50 N1 = 1.1 microM, IC50 N8 = 2.4 microM). In contrast, similar pretreatment with DFMO or coincubation with spermidine had no effect on the cytotoxicity induced by the aziridine-containing alkylating agent, N,N',N"-triethylenethiophosphoramide (thiotepa) (IC50 = 2.4 microM). The cytotoxicity induced by 24-h incubation with either N1- or N8-aziridinyl spermidine was not altered by removal of those compounds and incubating treated cells in medium augmented with 3.7 microM spermidine. However, and as expected, similar maneuvers did not reverse the cell growth-inhibitory effect induced by 24-h incubation with 100 microM DFMO. Cellular accumulation of both N1- and N8-aziridinyl spermidine increased with increasing extracellular concentrations. N1-Aziridinyl spermidine was accumulated to a greater degree than was the N8-analogue, achieving up to 6-fold higher intracellular concentrations at the same extracellular concentration. Cellular accumulation of both aziridinyl compounds was greatly enhanced by 24-h pretreatment with DFMO. Both N1- and N8-aziridinyl spermidine inhibited the uptake of spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. These results imply that the cytotoxic mechanism of the aziridinyl spermidine analogues is, to a great extent, dependent on their polyamine nature and may imply selectivity for rapidly growing and neoplastic cells.
我们之前描述了2种多胺类似物的合成及其细胞毒性特性，在这2种类似物中，亚精胺的N1-或N8-氨基被烷基化部分氮丙啶取代。然而，这些亚精胺氮丙啶类似物抑制细胞生长的机制仍不清楚。因此，我们进行了以下研究：(a) 用二氟甲基鸟氨酸（DFMO）预处理并与外源性亚精胺共同孵育对亚精胺氮丙啶诱导的细胞毒性的影响；(b) 亚精胺氮丙啶诱导的细胞毒性的可逆性；(c) 细胞对N1-和N8-亚精胺氮丙啶的摄取以及DFMO对该过程的影响；(d) N1-和N8-亚精胺氮丙啶对细胞多胺池以及亚精胺细胞内积累的影响。这2种亚精胺氮丙啶衍生物诱导的细胞毒性 [抑制细胞生长或使放射性标记前体掺入三氯乙酸沉淀物质的量减少50% 所需的浓度（IC50）：N1 = 0.2 μM，IC50 N8 = 0.4 μM]，在用100 μM DFMO预处理L1210细胞24小时后增强（IC50 N1 = 0.05 μM，IC50 N8 = 0.15 μM），并通过与3.7 μM亚精胺共同孵育而被阻断（IC50 N1 = 1.1 μM，IC50 N8 = 2.4 μM）。相比之下，用DFMO进行类似预处理或与亚精胺共同孵育对含氮丙啶的烷基化剂N,N',N"-三乙烯硫代磷酰胺（噻替派）诱导的细胞毒性没有影响（IC50 = 2.4 μM）。用N1-或N8-亚精胺氮丙啶孵育24小时诱导的细胞毒性，在去除这些化合物并将处理后的细胞在添加有3.7 μM亚精胺的培养基中孵育后没有改变。然而，正如预期的那样，类似操作并没有逆转用100 μM DFMO孵育24小时诱导的细胞生长抑制作用。随着细胞外浓度增加，N1-和N8-亚精胺氮丙啶的细胞内积累均增加。N1-亚精胺氮丙啶的积累程度高于N8-类似物，在相同细胞外浓度下，细胞内浓度可达6倍之高。用DFMO预处理24小时可大大增强这2种氮丙啶化合物的细胞内积累。N1-和N8-亚精胺氮丙啶均以剂量依赖性方式抑制亚精胺的摄取。测试化合物对多胺生物化学的干扰表现为它们耗尽细胞内腐胺以及亚精胺和精胺的能力。这些结果表明，亚精胺氮丙啶类似物的细胞毒性机制在很大程度上取决于它们的多胺性质，并且可能意味着对快速生长和肿瘤细胞具有选择性。
